Method of producing l-lysine by fermentation

ABSTRACT

ARTIFICIALLY INDUCED MUTANTS OF BREVIBACTERIUM LACTOFERMENTUM, CORYNEBACTERIUM GLUTAMICUM, CORYNEBACTERIUM LILIUM, AND CORYNEBACTERIUM ACETOACIDOPHILUM WHICH RESIST FEEDBACK INHIBITION BY S-(2-AMINOETHYL)-CYSTEINE AND REQUIRE AT LEAST ONE SUPPLEMENTAL NUTRIENT WHICH IS HERINE, PROLINE, ANANINE, NICOTINAMIDE, NICOTINIC ACID, PANTOTHENIC ACID, THIAMINE, GUANIDINE, ADENINE, HYPOXANTHINE, OR VITAMIN B12 PRODUCE L-LYSINE BY FERMENTATION OF OTHERWISE CONVENTIONAL CULTURE MEDIA IN HIGHER YIELDS THAN THE PARENT STRAINS LACKING THE NUTRIENT REQUIREMENT.

United States Patent 3,825,472 METHOD OF PRODUCING L-LYSINE BY FERMENTATION Koji Kubota and Yasuhiko Yoshihara, Kawasaki, Hayao Hirakawa, Yokohama, Hirotaka Kamijo, Kawasaki, Shigeki Nosaki, Isehara, Fumihiro Yoshinaga, Fujisawa, and Shinji kumura and Hiroshi Okada, Tokyo, Japan, assignors t'o Ajinomoto Co., Inc., Tokyo, Japan No Drawing. Continuation-impart of abandoned application Ser. No. 353,635, Apr. 23, 1973. This application Oct. 2, 1973, Ser. No. 402,818 Claims priority, application Japan, Apr. 27, 1972,

47/42,527;' Aug. 18, 1972, 47/ 82,641; Sept. 19, 1972, 47/94,030; Nov. 30, 1972, 47/120,247

Int. Cl. C12d 1/00 US. Cl. 195-29 9 Claims ABSTRACT OF THE DISCLOSURE Artificially induced mutants of Brevibacterium lactofermentum, Corynebacterium glutamicum, Corynebacterium ,lilium, and Corynebacterium acetoacidophilum which resist feedback inhibition by S-(Z-aminoethyD-cysteine and require at least one supplemental nutrient which is herine, proline, alanine, nicotinamide, nicotinic acid, pantothenic acid, thiamine, guanidine, adenine, hypoxanthine, orvitamin B produce L-lysine by fermentation of otherwise conventional culture media in higher yields than the parent strains lacking the nutrient requirement.

This application is a continuation-in-part of our copending application, Ser. No. 353,635, filed on Apr. 23, 1973, and now abandoned.

This invention relates to the production of L-lysine, and particularly to a method of producing L-lysine by bacterial fermentation.

L-Lysine has been produced heretofore by fermentation relying on three types of bacteria. The first type requires for its growth amino acids related to the biosynthesis of L-lysine and is exemplified by a homoserine-requiring mutant of Micrococcus glutamicus disclosed in Pat. No. 2,979,439. The second type includes threonineor methionine-sensitive mutants and mutants whose growth is inhibited by low concentrations of threonine or methionine, but which require threonine for their growth (French Patents No. 1,533,688). The third type is characterized by resistance to S-(2-aminoethyl)-L-cysteine (AEC) which is a sulfur analogue of L-lysine, as disclosed in Pat. No. 3,707,441.

We now have found that L-lysine is produced at higher concentrations than were available heretofore under comparable conditions by mutants which combine resistance to feedback inhibition by lysine or lysine analogues with a requirement for at least one specific nutrient which is serine, proline, alanine, nictoinamide, nicotinic acid, pantothenic acid, thiamine, guanidine, adenine, hypoxanthine, or vitamin B when these mutants are cultured on otherwise conventional media including sources of assimilable carbon and nitrogen, inorganic ions, and unspecific organic growth promoting agents.

The mutants of the invention are derived by means of conventional mutagenic agents from parent strains of the genera Brevibacterium and Corynebacterium, and by screening of the mutants so produced for the necessary resistance to feedback inhibition by lysine and its analogues, such as AEC in concentrations of 1 mg./ml. or more, and a requirement for one of the nutrients enumerated above. Because of their resistance to lysine analogues, the mutants of the invention produce lysine at a predictable rate and in predictable amounts without being seriously affected by variations in the amounts 3,825,472 Patented July 23., 1974 of threonine and :other organic nutrients in the fermentation medium employed.

The latter thus may be a natural nutrient medium or .a synthetic nutrient medium providing the basic needs of the microorganisms of the invention together with the specific organic nutrient or nutrients of the group enumerated above.

Suitable carbon sources include theusual carbohydrates, such as glucose, fructose, maltose, sucrose, starch, starch hydrolyzate, molasses, also organic acids, particularly acetic acid, propionic acid, or fumaric acid, and alcohols, particularly methanol and ethanol, and a medium may contain several different carbon sources. Some of the new mutants assimilate hydrocarbons as a major or minor carbon source.

Nitrogen may be supplied by ammonium salts, nitrates, urea, amino acids, cornsteep liquor, yeast extract, meat extract, fish meal, peptone, bouillon, casein hydrolyzates, and mixtures thereof, and also by ammonia.

Necessary inorganic ions may be provided by magnesium sulfate, sodium phosphate, potassium monoand dihydrogen phosphate, iron sulfate, manganese chloride, calcium chloride, sodium chloride and others.

The specific nutrients required by the mutants of the invention and enumerated above may be provided in the form of natural products containing the same, and simple derivatives of natural products, such as soybean protein hydrolyzate, yeast extract, cornsteep liquor, peptone, casein hydrolyzate, and the like. The specific nutrients must be held at a relatively low concentration, generally below the concentration producing optimum growth, and to be determined experimentally for each nutrient and each mutant strain, the phenomenon being well known in itself.

The fermentation is carried out in a temperature range between 24 and 37 C., and maximum lysine concentrations are generally reached after 24 to 96 hours of culturing under aerobic conditions maintained by agitation and/or aeration. The pH value of the medium should be controlled between 5 and 9 by additions of calcium carbonate, ammonia, organic or inorganic acids, as may be required.

Conventional methods may be used for recovering the accumulated lysine from the culture broth, preferably after removal of the microbial cells by filtration or centrifuging. Cation exchange resins conveniently absorb lysine fi-om the cell free broth. The lysine concentration in the culture broth may be monitored by means of lysine decarboxylase, and the product is readily identified by paper chromatography.

The following Examples are further illustrative of this invention.

ice

EXAMPLE 1 Cells of Brevibacterium lactofermentum ATCC 13869 were irradiated with X-rays to induce mutation, and cultured thereafter in a complete agar fiat plate at 31 C. for 4-l0 days. Four mutant strains were isolated by the replica method from colonies apearing on the plate according to specific nutrient requirements for serine, pantothenic acid, thiamine, and guanine or adenine, which were determined by standard methods known in the art.

The four strains so obtained were each treated with 200 ,ug/ml. nitrosoguanidine at 30 C. for 30 minutes, and inoculated on agar flat plates containing 2 g./d1. glucose, 0.3 g./dl. urea, 0.5 g./dl. threonine, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 ,ug./l. biotin, 100 ,ug./l. thiamine, 500 ug/ml. ABC, 2 g./dl. agar, and, respectively, 30 mg./dl. serine, 10 mg./l. calcium pantothenate, and 100 mg./l. adenine for the three strains requiring these nutrients.

The four cultures were adjusted to pH 7.0 and cultured at 31 C. for 4 to 10 days. The colonies appearing on the plates were tested for their lysine-producing ability and-the .following four strainswere isolated:

B. lactofermentum Y-108 (ATCC 21798; FERM P-l443) B. lactoferme'ntum AC-73 (ATCC 21799; FERM P-1444) B. lactofermentum AD- (ATCC 21800; FERM P-1445 B. lactofermentum AD-162 (ATCC 21801; FERM P- The parent strain B. lactofermentum ATCC 13869 and the four mutants were cultured 24 hours at 31 C. with shaking on a minimal medium of 2 g./dl. glucose, 1 g./dl. ammonium sulfate, 0.3 g./dl. urea, 0.1 g./dl. KH PO 0.04 g. /dl. MgSO .7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 pg/l. biotin, and 200 g/l. thiamine hydrochloride. In parallel control tests, the four mutants were grown on respective modified media ditfering from the minimal medium by an addition of 30 mg./dl. serine (Y-108), an addition of mg./l. calcium pantothenate (AC-73), the absence of thiamine hydrochloride (AD-5), and the addition of 100 mg./l. adenine (AD-162).

Eachcultured broth was diluted with 25 volumes of water, and its optical density was determined as a measure of growth by measuring light absorbancy at 562 mp. The results are listed in Table 1.

TABLE 1 Optical density Brevibacterium lactofermentum Minimal medium Modified medium ATCC 13869 0. 400 Y-lO 0. 065 0. 410 110-73 0. 059 0. 405

-AD5 0. 380 0. 075 AD-162 0. 052 375 EXAMPLE 2 B. lactofermentum ATCC 13869 was treated with 250 ug/ml. nitrosoguanidine at 30 C. for 30 minutes, inoculated on an agar plate containing 2 g./dl. glucose, 0.3 g./ d1. urea, 1 g./dl. ammonium sulfate, 0.5 g./ d1. threonine,

vOil g./dl. KH PO 0.04 g./dl. MgSO .7H O, 2 p.p.m.

Fe++, 2 p.p.m. Mn++, 50 ,ug./l. biotin, 200 [Lg./l. thiamine.HCl, 5 mg./ml. ABC, and 2 g./dl. agar, at pH 7.0. After 4 to 10 days culturing at 31 C., AEC resistant strains were isolated from the colonies appearing on the cultured agar plate, and mutants capable of producing L-lysine were selected. They had no specific nutrient requirements and were further subjected to nitrosogunidine treatment as above, and the following six mutants resistant to ABC and capable of producing lysine in high yields were isolated:

B. lactofermentum AJ-3391 (FER'M P-1570) B. lactofermentum AJ-3392 (PERM P-1571) B. lactofermentum AJ-3393 (PERM P-l572) B. lactofermentumAI-3394 (PERM P-1573) B. lactofermentum AJ-33995 (FERM P-1574) B. lactofermentum All-3396 (FERM P-l575) 7 TABLE 2 Optical density Brevibacterium lactofermentum Minimal medium Modified medium EXAMPLE 3 Mutation was induced in B. lactofermentum' ATCC 13869 as in Example 2, and an ABC-resistant mutant strain N0. 872, capable of producing lysine, was isolated from colonies growing on an agar plate differing from that employed in Example 2 by containing 0.4 g./dl. threonine, g/l. thiamine.HCl, and 4 mg./ml. AEC. Strain N0. 872, which does not require specific nutrients, was subjected to the same mutagenic treatment with nitrosoguanidine, and two mutants combining AEC-resistance with specific nutrient requirements, and capable of producing much L-lysine were isolated:

B. lactofermentum AI-3424 (FERM P-1711) B. lactofermentum AJ-3425 (PERM P-1712) Mutagenic treatment of B. lactofermentum AI-3424 further produced the lysine producing strain B. lactofermentum AJ-3429 (FERM P-1857).

The nutrient requirements of these three strains were determined or confirmed by inoculation of a minimal medium differing from that described in Example 1 by containing 0.05% NaCl and of modified media additionally containing amino acids and mixtures of amino acids in amounts of 50 mg./dl. per amino acid and/or nicotinamide or nicotinic acid in an amount of 5 mg./l. Optical densities of diluted culture broths were determined as a measure of growth as in the preceding Examples after 24 hours of culturing at 31 C.

The values listed in Table 3 are averages of two runs, and the amino acids identified by their abbreviated names are the naturally occurring L-isomorphs except for D-alanine, specifically identified as D-Ala. Nicotinamide and nicotinic acid are interchangeable and jointly identified as Nic..

TABLE 3 Optical density Modifying nutrient 5-3425 0 Ala. +Leu. +Nic. +Thr. +Met EXAMPLE 4 Corynebucterium glutamz'cum All-3463 (PERM P- 1987) which resists AEC but has no specific nutrient requirements was derived from Microcaccus gluramicus or Corynebacterium glutamicum ATCC 13032 by the mutation technique described in Example 3, and further mutation of strain AJ-3463 by the same technique produced C. glutamicum AJ-3458 (FERM P-1982), AJ-3460 (FERM P-1984), and ALI-3461 (FERM P-l985) which resist AEC and respectively require proline, guanine, and vitamin B +1eucine, as is evident from the following tests of their growth on a medium containing 2 g./dl. glucose, 1 g./dl. ammonium sulfate, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, 0.05 g./dl. NaCl, 50 gJl.

C'orynebacterium qlutamicum EXAMPLE- 5 Using the procedure described in Example 3, Corynebacterium glutamicum AJ-3397 (FERM P-l6l3) and Corynebacterium lilium AI-3464 (PERM P-2026) were obtained by mutation from Micrococcus glutamicus ATCC 13032 and Corynebacterium lilium NRRL B-2243 (ATCC 15990) respectively whose taxonomical features have been described in Pat. No. 3,087,863. The new .mutants resist ABC and respectively require serine and alanine.

Similarly, Corynebacteriunt acetoacidophilum ATCC 13870 yielded a mutant strain C. acetoacidophilum AJ 3465 (FERM P-.2027) by the method of Example 3. It resists ABC and requires alanine for its growth. Corynebacterium glutamicum AJ-3463 (FERM P-1987) was derived in the same manner fromC. glutamicum ATCC 13032, resists AEC, but does not require specific nutrients for its growth.

' The mutant strains produced in the manner of Examples 1 to 5 were employed in the production of lysine 'as describ'ed in the following additional Examples.

The soybean protein hydrolyzate described hereinafter contained 7% total nitrogen, and, per milliliter, 26 mg. serine, 30 mg. proline, 45 mg. leucine, 0.08 mg. nicotinamide, and 0.002 mg. vitamin B The yeast extract mentioned in the Examples contained 26 mg./g. guanine.

EXAMPLE 6 was further enriched, as indicated below, placed in a 50.0. ml. shaking flask, and sterilized. The batches were ,inoculatedrespectively with the four strains of B. lactfermentum derived from strain ATCC 13869 according to Example 1. In addition to the basic ingredients listed above,the medium'for strain AC-73 contained 0.1 mg./

ml. methionine and mg./ml. calcium pantothenate, and the medium for AD162 contained 100 mg./l. adenine and-100" mg./1. guanidine.

After 72 hours of cultivation with shaking at 31 C., the amounts of L-lysine, as hydrochloride, accumulated in the broths were 4.1 g. for strain Y-l08, 2.5 g./dl. for strain AC-73, 3.0 g./dl. for strain AD-S, and 2.8 g./dl. for strain AD-162.

The cultured broth of strain Y-108 was centrifuged to .remove the cells, and one liter of the supernatant was stripped of L-lysine by passage over a column of a cation exchange resin (Amberlite IR-120, H-type). The absorbed L-lysine was eluted with 3% aqueous ammonia, and the eluate was partly evaporated in a vacuum. Hydrochloric acid was added to the concentrate which was Minimal medium Modified medium then placed in an icebox to precipitate crystalline L-lysine hydrochloride dihydrate in an amount of 30.2 g.

EXAMPLE 7 The four strains of B. lacfofermentum. referred to in Example 6 were inoculated on respective seed culture media each containing 15 g./dl. glucose, 0.3 g./dl. ammonium acetate, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 ,ug./1. biotin, 200 g./l. thiamine hydrochloride, 1.5 ml./dl. soybean protein hydrolyzate, and 0.5 g./dl. yeast extract, and having a pH of 8.0. The seed cultures were held at 31 C. for 16 hours with stirring and aeration.

Respective 15 ml. inocula of the seed cultures were added to 300 ml. batches of fermentation media containing 3 g./dl. glucose, 0.5 g./dl. ammonium acetate, 0.2 g./dl. urea, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 ,ug./l. biotin, 50 ,ug./ l. thiamine.HCl, 3 mL/dl. soybean protein hydrolyzate, at pH 7.5. The media for B. lactofermentum AC-73 additionally contained 5 mg./l. calcium pantothenate, and those for B. lactofermentum AD-162 mg./l. each of adenine and guanine.

The fermentation mixtures were stirred at 1500 r.p.m. at 31.5 C. and aerated with one volume of air per minute and volume of broth. The pH of each medium was held automatically between 7.2 and 8.0 by additions of a 60% solution of acetic acid containing 0.25 mole ammonium acetate per mole of acetic acid. After 48 hours, it was found that strain Y-108 has produced 6.4 g./dl.

I lysine, strain AC-73 4.8 g./dl., strain AD-5 5.7 g./dI.,

and strain AD-162 6.0 g./ d1.

EXAMPLE 8 The four strains of B. lactofermentum referred to in Examples 6 and 7 were cultured at 31.5 C. for 16 hours with shaking on an aqueous seed culture medium containing 1.5 g./dl. glucose, 0.3 g./dl. urea, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, .2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 ,ug./l. biotin, 200 pg/l. thiamine hydrochloride, 1.5 mL/dl. soybean protein hydrolyzate, and 0.5 g./dl. yeast extract, and having a pH of 8.0.

15 m1. Inocula of each seed culture were transferred to 300 ml. batches of a fermentation medium containing 1 g./dl. glucose, 1.5 g./dl. ethanol, 0.5 g./dl. ammonium sulfate, 0.2 g./dl. urea, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7I-I O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 gJl. biotin, 50 ,ug./l. thiamine hydrochloride, 3 m1./dl. soybean protein hydrolyzate, and having an initial pH of 7.5. The media for strains AC-73 and AD-162 were supplemented as in Example 7.

Each fermentation mixture was stirred at 1500 r.p.m. and 31.5 C. for 48 hours while being aerated with an equal volume of air per minute. The pH was held at 7.0 to 7.5 by means of gaseous ammonia. The ethanol content of each medium was determined by gas chromatography, and the ethanol was replenished whenever residual ethanol fell to about 0.3 g./dl.

L-Lysine hydrochloride was accumulated by strain Y-l08 in an amount of 5.5 g./dl., by AC-73 in an amount of 4.4 g./dl., by strain AD-5 in an amount of 5.5 g./dl., and by strain AD-162 in an amount of 5.7 g./dl.

EXAMPLE 9 The six strains of B. lactofermenmm' derived from B. lactofermentum ATCC 13869 by the method of Example 2 were cultured on the basic medium of Example 6 which was enriched with soybean protein hydrolyzate at the rate of 3 mL/dl. for Ali-3391, AJ-3394, and AJ3396, 1.5 ml./dl. for AJ-3392 and All-3393, and 2 mL/dl. for AJ-3395, and with 5 mg. hypoxanthine per ml. for strain AJ-3396. Strains AI-3391 to All-3396 produced, in the same order, 2.9, 5.0, 4.6, 3.5, 3.1, and 3.0 g./dl. L-lysine (as the hydrochloride) in 72 hours.

7 EXAMPLE 10 The six strains of B. lactofermentum referred to in Example 9 were cultured on a medium containing acetate ions as the principal carbon source in the manner de scribed in Example 7, the medium of Example 7 being supplemented by mg./ml. hypoxanthine for strain AJ- 3396. The lysine concentrations (as lysine hydrochloride) accumulated after 48 hours incubation by the six strains AJ-3391 to AJ-3396 were, in the same order, 5.4, 7.1, 7.0, 6.5, 5.8, and 6.1 g./dl.

The amount of acetic acid consumed in the culture of B. lactofermentum AJ-3392 during the 48 hour period amounted to 30% of the initial culture volume, and 56.3 g. L-lysine hydrochloride dihydrate was recovered from one liter of the cell-free culture broth in the manner described in Example 6.

EXAMPLE 11 The fermentation medium described in Example 8, in which ethanol constitutes the principal carbon source, was used as a substrate for the six strains of B. lactofermentum referred to in Examples 9 and 10. The medium for strain AJ3396 was supplemented by 5 mg./dl. hypoxanthine. The amounts of lysine (as lysine hydrochloride) accumulated in the several broths after 48 hours were, in the same order as in the preceding Examples, 5.2, 6.3, 6.5, 6.1, 5.0, and 5.5 g./dl.

In the culture of B. lactofermenum AJ-3392, the L-lysine formed corresponded to 27.7% of the ethanol used, and 50.4 g. L-lysine hydrochloride dihydrate was recovered from one liter of the cell-free broth in the manner of Example 6.

EXAMPLE 12 B. lactofermentum AJ-3424, AI-3425 and the parent strain No. 872 were cultured as described in Example 6, but 200 ,ug./l. thiamine hydrochloride was added to the basic culture medium, and the medium for strains AJ- 3424 and No. 872 further was enriched with 1.5 ml./dl. soybean protein hydrolyzate, and 3 mL/dl. soybean protein hydrolyzate was added to the medium for strain A]- 3425.

After 72 hours of culturing, the amounts of L-lysine produced (as the hydrochloride) and the conversion rate of glucose to lysine were respectively 4.4 g./dl. (44%) for AI-3424, 3.2 g./dl. (32%) for AI3425, and only 1.8 g./dl. (18%) for the parent strain No. 872.

EXAMPLE 13 Seed cultures of the three strains referred to in Example 12 were prepared on a medium containing 1.5 g./dl. glucose, 0.3 g./dl. ammonium acetate, 0.1 g./dl. urea, 0.1 g./ld. KH PO 0.04 g./ld. MgSO .7H O, 2 p.p.m., Fe++, 2 p.p.m. Mn++, 50 ag/l. biotin, 200 ,ug./l. thiamine hydrochloride, and 2 ml./dl. soybean protein hydrolyzate, at pH 7.5. After 18 hours of culturing at 31 C. with stirring and aerating, 15 ml. inocula of the seed cultures were transferred to 300 ml. batches of sterilized fermentation medium in one liter jar fermentors. Each fermentation medium contained 2 g./dl. glucose, 0.5 g./dl. ammonium acetate, 0.2 g./dl. urea, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 ,ug./l. biotin, 50 ,ug./l. thiamine hydrochloride, and 2.5 ml./dl. soybean protein hydrolyzate, and had a pH of 7.5.

Each fermentation mixture was cultured at 3133 C. with stirring at 1500 r.p.m. and aeration at a rate of one volume of air per volume of culture medium per minute. The pH of each medium was held at 7.2 to 8.0 by periodic, automatic additions of a 60% acetic acid solution containing 0.25 mole ammonium acetate per mole acetic acid.

The lysine concentration achieved after 55 hours (as hydrochloride) was 7.2 g./dl. for strain AJ-3424, 6.1 g./ dl. for AJ-3425, and 4.0 g./dl. for strain No. 872. The amounts of acetic acid consumed were 22%, 24%, and 32% respectively.

8 3 EXAMPLE 14 The ammonium acetate in the seed culture medium of Example 13 was replaced by 0.5% ethanol, and 0.3 g./dl. urea was added. B. lactofermentum AJ-3424, AI 3425, and No. 872 were cultured on the modified seed culture medium at 31' C. for 18 hours with stirring and aeration;

A fermentation medium was prepared as in Example 8', but the ammonium acetate was replaced by 1 g./dl. glucose, 1 g./dl. ethanol, and 0.5 g./dl. ammonium sulfate. Respective batches of the fermentation medium were inoculated with the seed cultures and held at'pH 7.2-8.2 by introduction of gaseous ammonia. Ethanol was replenished when the amount of residual ethanol in a fermentation medium, as determined by gas chromatography, fell to about 0.3%.

The amounts of L-lysine hydrochloride accumulated in each fermentation medium after 48 hours were 6.6 g./dl. for AJ-3424, 5.6 g./dl. for AJ-3425, and 3.6 g./dl. for No. 872. Based on the ethanol used, the yields were 26%, 20%, and 17% respectively.

EXAMPLE 15 B. lactofermentum AI-3429 was cultured on a bouillon slant and then inoculated on 20 ml. batches of a fer.- mentation medium sterilized in 500 ml. shaking flasks at C. for 5 minutes. The medium contained 10 g./dl. glucose, 4.5 g./dl. ammonium sulfate, 0.1 g./dl. KH PO 0.04 g./dl. MgSO .7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn t, 50 ,LLg./l. biotin, 200 ag/l. thiamine hydrochloride, 1;ml./ dl. soybean protein hydrolyzate, and 5 g./dl. CaCO and had an initial pH of 7.0.

After 72 hours of fermentation at 31 C., the broth contained 5.1 g./dl. L-lysine (as the hydrochloride) corresponding to a yield of 51% based on the glucose initially present.

EXAMPLE 16 C. glutwmicum AJ-3397, AIL-3458, AJ-3460, AJ-3461; C. lilium AJ-3464, and C. acetoacidophilum AJ-3465 were cultured as in Example 15, except for an additional 0.5% yeast extract in the medium for C. glutamicum AJ- 3460. C. glutamicum AJ-3463 was cultured in the same manner as a control which lacks a nutrient requirement.

In the order in which the microorganisms are listed in the preceding paragraph, they produced 3.0, 3.0, 4.1, 3.1, 2.9, 3.2, and 2.0 g./dl. L-lysine (as hydrochloride). On the basis of 10 g./dl. glucose in the initial medium, 3.0 g./dl. lysine correspond to a yield of 30% EXAMPLE 17 The six microorganisms of the invention mentioned in Example 16 were cultured in a medium containing acetate ions as the principal carbon source, substantially as described in Example 7, but with an additional 0.5% yeast extract in the seed culture medium whose pH was adjusted to 7.0. The fermentation medium contained an additional 2.5 mL/dl. soybean protein hydrolyzate. I

After 55 hours of cultivation, the media onwhich the six microorganisms had been cultured contained, in the order indicated in Example 16, 5.0, 5.2, 4.7, 6.4, 5.5, and 5.8 g./dl. lysine (as the hydrochloride), and the acetic acid consumption was, in the same order, 22%, 23.5% 25%, 23%, 24%, and 22%.

EXAMPLE 18 C. glwtamicum AJ-3397, C. lilium Alf-3464, and C. acetoacidop-hilum AI-3465 were inoculated on respective batches of a seed culture medium and cultured at 31 C. for 18 hours with stirring and aeration. The medium contained 1.5 g./dl. glucose, 0.3 g./dl. urea, 0.1 g./dl. KH PO 0.04 g./dl. MgSO -7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 g./l. biotin, 200 ugJl. thiamine hydrochloride, and 1.5 mL/dl. soybean protein hydrolyzate. It had an initial pH of 7 .5.

300 ml. Batches of a fermentation medium were placed in one liter glass jar fermentors, sterilized, and inoculated with respective ml. portions of the seed cultures. The fermentation medium contained 1 g./dl. glucose, 1 g./dl. ethanol, 0.5 g./dl. ammonium sulfate, 0.2 g./dl. urea, 0.1 g./d1. KH PO 0.04 g./dl. MgSO -7H O, 2 p.p.m. Fe++, 2 p.p.m. Mn++, 50 ,LLg./1. biotin, 50 ,ug./l. thiamine hydrochloride, and 2.5 ml./dl. soybean protein hydrolyzate, and had an initial pH of 7.5.

Each fermentation batch was cultured at 31 to 33 C. with stirring and aeration at a rate of one volume of air per minute and volume of broth. A pH of 7.2 to 7.8 was maintained by introducing gaseous ammonia. Ethanol was replenished whenever the residual ethanol in a medium, as determined by gas chromatography, dropped to about 0.3%.

After 48 hours of cultivation, the three microorganisms had produced 4.8, 4.1, and 4.6 g./dl. L-lysine respectively (as hydrochloride) and had converted ethanol into L- lysine at respective yields of 23%, 20.5%, and 24%.

The microorganisms identified in this specification and the appended claims by accession numbers preceded by ATCC are freely available from the American Type Culture Collection in Rockville, Maryland, and those identified by accession numbers preceded by FERM P are equally available from the Fermentation Research Institute, Agency of Industrial Science and Technology, the Ministry of Industrial Trade and Industry, Chiba-shi, Japan.

What is claimed is:

1. A method of producing L-lysine which comprises:

(a) culturing a lysine-producing microorganism of genus Brevibacterium or Corynebacterium under aerobic conditions in an aqueous medium containing sources of assimilable carbon and nitrogen, inorganic salts, and minor organic nutrients necessary for the growth of said microorganism, at pH 5 to 9 until lysine accumulates in said medium,

(1) said microorganism resisting feedback inhibition by lysine and lysine analogues, and (2) said microorganism requiring at least one minor organic nutrient selected from the group 5. A method as set forth in claim 4, wherein said strain I is Brevibacterium lactofermentum ATCC 21798, ATCC 21799, ATCC 21800, ATCC 21801, FERM P-1570, FERM P-1571, FERM P-1572, PERM P-1573, FERM P-1574, FERM P1575, FERM P-171l, FERM P-1712, or FERM P-1857.

6. A method as set forth inclaim 3, wherein said microorganism is a strain of Corynebacterium glutamicum.

7. A method as set forth in claim 6, wherein said strain is Corynebacterium glutamicum FERM P-1613, FERM P-1982, FERM P-1984, or FERM P4985.

8. A method as set forth in claim 3, wherein said mi croorganism is Corynebacterium lz'lzum FERM P-2026.

9. A method as set forth in claim 3, wherein said microorganism is Corynebacterium acetoacidophilum FERM P-2027.

References Cited UNITED STATES PATENTS 3,708,395 1/1973 Nakayama et al. -29 3,707,441 12/1972 Shiio et a1 195-28 R 3,711,374 1/1973 Tanaka et a1 195-28 R A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner US. Cl. X.R. 195-28, 30, 47 

